Successful treatment of renal allograft rejection with a humanized antilymphocyte monoclonal antibody.
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Adult↗
Biomedical subjects
D T Harris
Publications and source records attributed to D T Harris.
Introduction: adjuvant and neoadjuvant therapy.
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Chemotherapy, Adjuvant↗
Adjuvant systemic therapy for breast cancer.
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Antineoplastic Combined Chemotherapy Protocols↗
Target-cell sensitivity to natural killer-cell lysis is determined by the expression of a novel antigen in conjunction with major histocompatibility complex class-I molecules.
Recently, a panel of monoclonal antibodies (MoAbs) was developed that identified a novel tumour-cell antigen conserved across species (mouse, rat and man). Fluorescence-activated cell sorter (FACS) analysis demonstrated that this antigen was expressed at highest levels on human tumour cell lines sensitive to natural killer (NK)-cell lysis. These MoAbs inhibited NK cell lysis of K562 target cells (by up to 90%), as well as a variety of other NK-sensitive target cells. Biochemical analyses revealed that the MoAbs reacted with a polypeptide of 42 kDaltons, distinct from other known cell surface antigens. Now the expression of this antigen has been analysed further with a panel of 24 tumour cell lines to determine its role in NK cell function. The expression of target cell major histocompatibility complex (MHC) molecules in conjunction with sensitivity to NK cell lysis was examined also. For each of the 24 cell lines it was found that the level of expression of the novel target cell antigen determined the sensitivity of the cell line to NK cell lysis. However, the level of MHC antigen expression could modulate target cell sensitivity to NK cell lysis, in that high levels of MHC class-I molecule expression resulted in a target cell that was insensitive to NK cell lysis regardless of the level of expression of the novel antigen. Thus, for most transformed cell lines, sensitivity to NK cell lysis appeared to be determined by the expression and levels of the novel antigen in association with MHC class-I molecules.
Adolescent↗
Evolutionary conservation of a human function-associated molecule on murine natural killer cells: expression and function.
Using a novel anti-natural killer (NK) cell monoclonal antibody (MoAb), we have recently identified an evolutionary conserved function-associated molecule (FAM) present on fish, rat and human NK cells. This molecule is involved in NK cell function as anti-FAM MoAbs inhibit cytotoxicity, stimulate lymphokine secretion and inhibit conjugate formation between effector cells and target cells. We now have examined murine NK cells for the presence of this structure. It was observed by two-colour flow cytometric analysis that the anti-FAM MoAb 5C6 specifically bound to a subpopulation of nylon wool non-adherent splenic lymphocytes (19-20%). The expression of the FAM molecule was restricted to NK cells that expressed the NK1.1 antigen. Neither T cells, B cells, nor macrophages reacted with the anti-FAM MoAb. Analysis of FAM expression in various lymphoid tissues revealed that splenocytes expressed the greatest numbers of MoAb(+) cells. Generation of lymphokine-activated killer (LAK) cells and adherent lymphokine-activated killer (ALAK) cells resulted in higher levels of FAM expression. The anti-FAM MoAb 5C6 also detected the presence of FAM on fresh SCID NK cells. It was demonstrated that the anti-FAM MoAb 5C6 inhibited the lysis of target cells by endogenous NK cells, activated NK cells, 5d LAK cells, ALAK cells and SCID NK cells. Moreover, conjugate assays demonstrated involvement of this molecule in recognition between NK cells and target cells.
Animals↗
Cord blood transplantation: implications for graft vs. host disease and graft vs. leukemia.
Cord blood (CB) was analyzed for its alloreactive immune potential to evaluate its capacity to mediate graft vs. host disease (GVHD) and graft vs. leukemia (GVL) effects. Cord blood was observed to display minimal innate cytotoxic capacity but was capable of rapidly developing significant nonspecific natural killer cell-like effector mechanisms. Cord blood was unable to generate effective alloantigen-specific cytotoxic T lymphocytes (CTL), which was at least partially due to an altered lymphokine profile. The frequency of alloreactive T cells present in CB (whether assessed as total responding T-cell or CTL precursors) was greatly reduced as compared to adult peripheral blood lymphocytes (PBLs). However, the frequency of nonspecific effector cells was equivalent to PBLs. Significantly, CB T cells seemed to have undergone some type of developmental tolerance to maternal HLA antigens in utero, which could greatly increase the utility of CB in familial transplants. That is, CB T cells were unresponsive to noninherited maternal HLA antigens. Finally, CB demonstrated significant GVL capacity whether measured in vitro or in an animal model in vivo. Thus, the use of CB in most transplant settings should be free of the immunological problems associated with GVHD yet still be an effective mediator of GVL.
Cytotoxicity, Immunologic↗
Analysis of the alloreactive capacity of human umbilical cord blood: implications for graft-versus-host disease.
Hematopoietic reconstitution by bone marrow transplantation (BMT) is used as therapy for the treatment of various malignancies and genetic blood disorders. Allogeneic BMT is the most common application of this treatment but is frequently associated with graft-versus-host disease (GVHD). Recent clinical studies have shown that sibling transplant using umbilical cord blood (UCB) is an acceptable alternative to BMT and may involve fewer problems with GVHD. We have investigated the in vitro alloreactive capacity of UCB as it relates to allogeneic transplantation. Initial screening assays demonstrated that UCB T cells were functionally immature. It was not possible to generate significant levels of alloantigen-specific cytotoxic T lymphocytes (CTL) in either primary or secondary mixed lymphocyte cultures. Limiting dilution analyses revealed that cord blood T cells were 10-1000 x less alloreactive in terms of proliferative T cells (PTLp) and cytotoxic T cells (CTLp) compared with adult peripheral blood lymphocytes (PBL). However, UCB was equivalent to adult PBL in terms of natural killer (NK) and lymphokine-activated killer (LAK) cell precursors. Analysis of cells from alloantigen-stimulated MLC revealed that UCB generated primarily CD4+ and CD16+ cells that made little or no IL-4, IL-6, TNF-alpha or IFN-gamma on antigenic stimulation. Cold target inhibition analyses revealed that alloantigen-stimulated cord blood T cells had a fine specificity similar to NK cells. From these in vitro results cord blood would seem to be unlikely to mediate severe GVHD reactions in vivo and should be suitable for allogeneic transplantation.
Adult↗
Immunoreactivity of umbilical cord blood and post-partum maternal peripheral blood with regard to HLA-haploidentical transplantation.
Recent clinical reports have demonstrated that umbilical cord blood (CB) may be utilized as a source of transplantable hematopoietic stem cells when bone marrow is not available. However, it is not apparent that CB can be used to transplant partially HLA-matched siblings or matched, non-familial recipients. In this study the immunoreactivity of CB has been investigated within familial confines; 14 families were analyzed at the time of birth of their child and six of these families were reassessed at 6 months post-partum. In mixed lymphocyte reactions, CB was unable to significantly respond to stimulation with cells from either the mother or father. Furthermore, unlike adult peripheral blood, CB displayed depressed immune responses to alloantigen and T cell mitogen. At 6 months, the immune responses of the infant demonstrated normal development in terms of alloantigen and mitogen responses. However, at 6 months both the mother and the infant demonstrated a continued immune tolerance to one another. The data suggest that CB could be used in familial transplant situations when siblings are HLA-haploidentical if the donor/recipients are chosen based on the paternal haplotype. Furthermore, maternal bone marrow harvested during the 6 months immediately following delivery of a child also should be suitable as a stem cell graft in haploidentical situations.
Cytotoxicity, Immunologic↗
Collection, separation and cryopreservation of umbilical cord blood for use in transplantation.
Bone marrow transplantation (BMT) is limited by the paucity of HLA-matched donors and the frequent occurrence of graft-versus-host disease (GVHD). Recent clinical reports have implied that the use of umbilical cord blood (UCB) may alleviate some of the problems associated with BMT. Banks of frozen UCB could make the problem of finding suitable stem cell donors easier and stem cell grafts would be more readily available. However, definitive experiments are needed to develop optimal methods for collection, separation and storage of cryopreserved UCB for extended periods of time. We have found that several simple techniques may be utilized to collect large volumes of UCB (up to 220 ml). Also, modification of a common density gradient separation method permits recovery of large quantities of UCB mononuclear cells. Finally, we have examined the effects of prolonged frozen storage on the ability to recover viable and functional UCB, particularly stem/progenitor cells. It was observed that storage of UCB in liquid nitrogen for as long as 7 years had minimal effects on cell viability, cellular composition of UCB and progenitor/stem cell capacity. Thus, the establishment of UCB banks for use in transplantation appears to be a feasible approach.
Blood Banks↗
Human bone marrow and umbilical cord blood cells generate CD4+ and CD8+ single-positive T cells in murine fetal thymus organ culture.
Murine fetal thymus lobes isolated from both normal and scid/scid mice can be colonized by donor cells from either human bone marrow or human umbilical cord blood in vitro. Subsequent organ culture results in a transient production of a few CD4+ CD8+ (double-positive) cells and then the accumulation of CD4+ or CD8+ (single-positive) T cells. A significant number of immature T-cell intermediates (e.g., CD8low, CD3-/low cells) were present in early organ cultures, suggesting that these were progenitors of the mature CD3+/high single-positive T cells that dominated late cultures. Depletion of mature T cells from the donor-cell populations did not affect their ability to colonize thymus lobes. However, colonization depended on the presence of CD7+ progenitor T cells. Limiting dilution experiments using mature T-cell populations (human peripheral blood leukocytes, human bone marrow cells, and human umbilical cord blood cells) suggested that thymic organ culture supports the growth of progenitor T cells but does not support the growth of mature human T cells. Each of these donor populations produced single-positive populations with different CD4/CD8 ratios, suggesting that precursor cells from different sources differ qualitatively in their capacity to differentiate into T cells.
Animals↗
Expression of an evolutionarily conserved function associated molecule on sheep, horse and cattle natural killer cells.
Natural killer (NK) cells are large granular lymphocytes that lyse a wide variety of transformed and virally-infected target cells without prior exposure to antigen, and without restriction by major histocompatibility complex antigens. Although NK cells have been identified in a variety of mammalian species, how NK cells recognize antigen and trigger lysis is unknown. Recently, monoclonal antibodies made against NK-like cells from teleost fish were shown to react with NK cells from humans and rats, and to inhibit their cytolytic activity. The role of this apparently evolutionarily conserved function-associated molecule (FAM) has been further investigated utilizing a variety of domesticated farm animal species. It was observed that the anti-FAM mAb reacted specifically with peripheral blood lymphocytes isolated from sheep, horses and cattle. Further, the anti-FAM mAb inhibited NK cell lytic activity in each of these species. Finally, the anti-FAM mAb was found to inhibit conjugate formation between NK and target cells, implying that the FAM was involved in antigen recognition by NK cells in each of these species. In conclusion, it appears that NK cell function is mediated by an evolutionarily conserved FAM in a wide variety of species.
Animals↗
Analysis of the hairless mouse as a model for the effects of aging on the immune system.
Studies into the effects of aging on the immune system are hampered by the lack of a suitable animal model that is readily available and cost efficient. The mutant mouse, hairless (hr/hr genotype), has been shown to undergo an accelerated thymic involution with accompanying immunodeficiency. Thus, this strain of mouse has been proposed as a model for studying the interactions of aging and immune function. We have investigated the effects of homozygous hr gene expression over time on the immune function of these mice. It was observed that homozygous hr gene expression had minimal effects on peripheral lymphocyte subset compositions but did appear to result in changes in thymic differentiation. Further, hr/hr mice displayed decreased proliferative responses to IL2 and mitogen stimulation, although cytotoxic responses (both NK and T cell mediated) appeared normal. These defects appear to be attributable to T helper cell dysfunction. Each of the changes found in hr/hr mice were distinct from those seen with age-matched control mice. Thus, the hr/hr inbred strain of mouse does not appear to be a suitable model for use in analyzing the effects of aging on the immune system.
Aging↗
A novel target cell antigen involved in the NK-like lytic activity of antigen-specific cytotoxic T lymphocytes.
Monoclonal antibodies (mAbs) have been derived which identify a target cell antigen involved in human natural killer (NK) cell lysis. The effects of the mAbs on the NK-like cytotoxic activity exhibited by different populations of human cytolytic T cells were examined. The anti-target cell mAbs 1E7 and 18C2 inhibited the lysis of K562 target cells by endogenous NK cells, antigen-specific cytotoxic T lymphocytes (CTL) with NK-like activity, and non-major histocompatibility complex (MHC)-restricted T cells. Cytolysis of K562 target cells was not affected by treatment of the target cells with the anti-class I HLA mAb W6/32. Further, the anti-target cell mAbs had no effect on antigen-specific lysis by the CTL. The mAb W6/32, however, inhibited the antigen-specific killing. Experiments at the single-cell level revealed that the anti-target cell mAbs inhibited the formation of conjugates between the effector cells and K562 tumor cells but had no effect on CTL binding to the antigen-specific target cells. Thus, antigen-specific CTL exhibiting NK-like lytic function appeared to recognize a novel target cell antigen that is distinct from typical MHC antigens and is identical to the target-cell antigen recognized by true NK cells.
Adolescent↗
Blastomycosis in transplant recipients: report of a case and review.
Fungi cause serious, often fatal infections in immunocompromised hosts. Recipients of solid organ and bone marrow transplants are predisposed to invasive fungal infections with Candida species, Aspergillus species, and Cryptococcus neoformans. In contrast, infections with Blastomyces dermatitidis have rarely been diagnosed in transplant recipients. We describe a patient who received an orthotopic heart transplant and developed recurrent disseminated blastomycosis. Other reported cases of blastomycosis in transplant recipients are summarized. Clinical presentation, treatment options, and morbidity associated with infections with B. dermatitidis in transplant patients are reviewed.
Amphotericin B↗
Identification of a vimentin-like function associated molecule (FAM) on rat NK cells: evidence for receptor function.
Monoclonal antibody (MoAb) 5C6 specifically binds to fish, rat and human NK cells and inhibits cytotoxicity. The molecule recognized by this MoAb is a 50-53-kDa membrane protein on rat leukaemic NK (CRC) cells. In the present study, we have obtained a partial internal amino acid sequence from a purified 42-kDa fragment of the CRC-function associated molecule (FAM). Three tryptic peptide fragments were sequenced and each showed homology to intermediate filament vimentin sequences as deduced from (GenBank) mouse cDNA sequences. Amino acid composition analysis indicated that similar to cytoskeletal vimentin, the FAM contained a high percentage of non-polar amino acids. To further assess the similarities between this protein and vimentin, two commercially available anti-vimentin MoAbs and one anti-vimentin polyclonal antibody were tested for binding and inhibition of NK cytotoxicity. All anti-vimentin MoAbs inhibited killing by rat NWNA cells of appropriate targets. Anti-vimentin MoAb 13.2 bound to 41% of NWNA cells compared with approximately 58% binding for MoAb 5C6. Capping and sequential binding experiments showed that MoAb 5C6 effectively removed, from CRC-cell membranes, the protein recognized by MoAb V9. Sequential addition of these two MoAbs (MoAb 13.2 followed by MoAb V9) to CRC cells did not produce competitive binding. Biochemical and Western blot analysis of the vimentin-like protein obtained from CRC cells indicated that this protein has a molecular weight of 48-50 kDa, with an isoelectric point of pH 6.1-6.3. This protein is cross-reactive by Western blot analysis with anti-vimentin and anti-intermediate filament (IFA) antigen MoAbs but not with anti-desmin or anti-actin MoAbs. The molecular weight heterogeneity (43 versus 48-50 kDa) of the CRC protein was also examined. Western blot analysis of the CRC extract after different in vitro incubation times at 37 degrees C and 4 degrees C demonstrated that the 50-53-kDa 'native' protein degraded to a 42-kDa protein by 24 and 48 h respectively. This degradation was inhibitable by 10 mM EGTA. Evidence is presented which indicates that a vimentin-like protein on transformed rat NK cells may be an antigen binding receptor which initiates target cell lysis.
Amino Acid Sequence↗
Prophylactic cranial irradiation in small-cell lung cancer.
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Brain↗
Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes.
Successful implementation of bone marrow transplantation for hematopoietic reconstitution is limited by the lack of suitably HLA-matched donors and by the occurrence of graft-versus-host disease that frequently accompanies this procedure. Recent clinical reports have implied that the use of umbilical cord blood as a source of transplantable stem cells may solve these problems. To date, definitive experiments have not been performed to assess the immunological potential of T cells found in umbilical cord blood, which could mediate graft-versus-host disease. In the present study we have observed that umbilical cord blood contains T lymphocytes that appear to be phenotypically immature. In addition, umbilical cord blood lymphocytes appeared to be functionally immature as shown by minimal responses to stimulation with interleukin 2, phytohemagglutinin, or alloantigens. Thus, umbilical cord blood may be more suitable for allogeneic transplantation than bone marrow in that these cord blood cells may not be as capable of mediating graft-versus-host disease.
Animals↗
Detection of function-associated molecules on rat NK cells and their role in target cell lysis.
Anti-effector cell mAb 5C6.10.4 (5C6) inhibits cytotoxic activity of fish nonspecific cytotoxic cells (NCC). We now show that 5C6 also inhibits mammalian NK cell activity using fresh and cultured (CRC) leukemic rat NK cells. The inhibitory activity of 5C6 was caused by blocking of conjugate formation between NK cells and YAC-1 targets. Binding studies done by flow cytometry (FCM) showed that mAb 5C6 specifically bound to 8% of unfractionated rat spleen cells. Enrichment by nylon-wool fractionation produced 27.2% specific binding, along with a 3.4-fold enrichment in cytotoxic activity. Tissue distribution studies revealed that the highest number of cells recognized by mAb 5C6 were found in NWNA spleen cells (28.7%), followed by liver (18.9%) and peripheral blood (13.9). Two-color FCM showed that although all 3.2.3 mAb-positive cells were also stained with mAb 5C6, a small percentage of 3.2.3. negative noncytotoxic NWNA spleen T cells were 5C6 positive. Redirected lysis experiments demonstrated that anti-effector mAb-producing myeloma cells could be killed by CRC and NWNA spleen cells. In addition, mAb 5C6 produced specific inhibition of redirected lysis of each myeloma target. Experiments were also conducted to determine the signaling capability of the FAM complex. Binding of the anti-FAM mAbs to NWNA rat spleen cells caused a rapid increase in cytosolic free calcium of approximately 472 nM. Western blot analysis of CRC cell lysates showed that the molecules recognized by anti-FAM mAbs have molecular weights of 38 and 42 kDa. These studies indicate that the anti-effector mAbs recognize a functionally relevant molecule on rat NK cells that is involved in the first steps of cytolysis, i.e., antigen recognition, and which also triggers the activation of signal-transducing events in these cells.
Animals↗